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Simple Summary The aquaculture sector provides for nearly half of the world’s seafood consumption, thanks to its large expansion over the last 30 years. Despite this intense growth, clear guidelines for responsible practices and animal wellbeing are lacking. Gene expression studies are a fundamental tool for understanding welfare, but stress-markers in aquaculture fish species are poorly studied. In addition, the biostatistical analysis of gene expression data is not trivial, and this study applies different statistical methods in order to evaluate potential differences in the gene expression levels between control fish and fish acutely stressed by exposure to air. Abstract In this study, a stress trial was conducted with common carp, one of the most important species in aquaculture worldwide, to identify relevant gene regulation pathways in different areas of the brain. Acute distress due to exposure to air significantly activated the expression of the immediate early gene c-fos in the telencephalon. In addition, evidence for regulation of the two corticotropin-releasing factor (crf) genes in relation to their binding protein (corticotropin-releasing hormone-binding protein, crh-bp) is presented in this preliminary study. Inferences on the effects of due to exposure to air were obtained by using point estimation, which allows the prediction of a single value. This constitutes the best description to date of the previously generally unknown effects of stress in different brain regions in carp. Furthermore, principal component analyses were performed to reveal possible regulation patterns in the different regions of the fish brain. In conclusion, these preliminary studies on gene regulation in the carp brain that has been influenced by exposure to a stressor reveal that a number of genes may be successfully used as markers for exposure to unfavourable conditions.

The first part of this study evaluates the occurrence of mycotoxin patterns in feedstuffs and fish feeds. Results were extrapolated from a large data pool derived from wheat (n = 857), corn (n = 725), soybean meal (n = 139) and fish feed (n = 44) samples in European countries and based on sample analyses by liquid chromatography/tandem mass spectrometry (LC-MS/MS) in the period between 2012–2019. Deoxynivalenol (DON) was readily present in corn (in 47% of the samples) > wheat (41%) > soybean meal (11%), and in aquafeeds (48%). Co-occurrence of mycotoxins was frequently observed in feedstuffs and aquafeed samples. For example, in corn, multi-mycotoxin occurrence was investigated by Spearman’s correlations and odd ratios, and both showed co-occurrence of DON with its acetylated forms (3-AcDON, 15-AcDON) as well as with zearalenone (ZEN). The second part of this study summarizes the existing knowledge on the effects of DON on farmed fish species and evaluates the risk of DON exposure in fish, based on data from in vivo studies. A meta-analytical approach aimed to estimate to which extent DON affects feed intake and growth performance in fish. Corn was identified as the ingredient with the highest risk of contamination with DON and its acetylated forms, which often cannot be detected by commonly used rapid detection methods in feed mills. Periodical state-of-the-art mycotoxin analyses are essential to detect the full spectrum of mycotoxins in fish feeds aimed to prevent detrimental effects on farmed fish and subsequent economic losses for fish farmers. Because levels below the stated regulatory limits can reduce feed intake and growth performance, our results show that the risk of DON contamination is underestimated in the aquaculture industry.

Periods of unfavorable storing conditions can lead to changes in the quality of fish feeds, as well as the development of relevant mycotoxins. In the present study, a commercial fish feed was stored under defined conditions for four weeks. The main findings indicate that even storing fish feeds under unsuitable conditions for a short duration leads to a deterioration in quality. Mycotoxin and fungal contamination were subsequently analyzed. These investigations confirmed that different storage conditions can influence the presence of fungi and mycotoxins on fish feed. Notably, ochratoxin A (OTA) was found in samples after warm (25 &deg, C) and humid (&gt, 60% relative humidity) treatment. This confirms the importance of this compound as a typical contaminant of fish feed and reveals how fast this mycotoxin can be formed in fish feed during storage.

Although several studies have reported co-occurrence of mycotoxins in fish feed limited information is available on the physiological effects. In the current study, the single and combined effect of added dietary AFB1 and ZEN in various concentrations for 60 days was tested in rainbow trout on growth parameters, mortality, intestinal digestive enzymes activity and on serum and intestinal oxidative stress and immunological parameters and on intestinal histopathology, versus untreated controls. Both mycotoxins groups irrespective of concentration, showed a significant deterioration of SGR and FCR as compared to control, whereas no consistent differences in mortality were present. The small intestinal enzyme activity was significantly reduced by ZEN in particular at the higher mycotoxin concentrations and when combined with AFB1. Serum and intestinal immunological parameters were significantly affected largely consistent with additive and synergistic negative effects on the (innate) immune system, in particular enhanced inflammation. The latter was also reflected in the serum and intestinal oxidative stress parameters. Histopathological examination showed decreasing villus length and Goblet cell density with increasing mycotoxin load. It is concluded that the combination of AFB1 and ZEN clearly increased the toxic effect on the fish as far as the serum and intestinal parameters are concerned. This is in some cases merely additive, but clear synergistic toxicity was seen too, in particular at the higher mycotoxin concentrations. Surprisingly, the increasing intestinal impairment did not translate into increased growth retardation and mortality since those were similar in all non-control groups. This may be consistent with anabolic effects of mycotoxin (metabolites) as has been described before.

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch), Mycotoxins are difficult to monitor continuously, and a tool to assess the risk would help to judge if there is a particular risk due to the inclusion of certain feed ingredients. For this, the toxin contents of 97 commercial fish feeds have been estimated, and the most prominent toxins in fish feed are calculated to be deoxynivalenol, zearalenone, fumonisins and enniatins. These pose a risk to fish well-being, as can be calculated by the Bayesian models for determining the critical concentrations 5% (CC5) for the different toxins. Besides fishmeal, wheat, soybean products and corn are regularly used as fish feed ingredients. The calculated scenarios show that fish are at high risk of toxin contamination if feed ingredients of low quality are chosen for feed production. Due to this, specific maximum allowable levels for several mycotoxins in fish feeds should be established.

Ethoxyquin (EQ) is an antioxidant that has, to date, been commonly used in feed production. Reports on the detrimental effects of this substance on vertebrates are growing, but effects in aquatic systems have rarely been described. Therefore, the present study was conducted using serial concentrations of EQ ranging from 0.03 to 16.5 mg l-1 to determine effects on 3 types of aquatic organisms. In zebrafish, 5 mg l-1 EQ caused mortality (25%) and a further 62.5% of the embryos showed yolk sac edema as well as deformed bodies or missing eyes. Furthermore, all the investigated EQ concentrations decreased the heart rate of the embryos. The lowest observed effect level was 0.31 mg l-1. In addition to zebrafish, the study also used water fleas Daphnia magna and green algae (Scenedesmus obliquus and Chlorella vulgaris). These treatments revealed that daphnids are also sensitive to EQ, exhibiting detrimental effects with a half-maximal effective concentration (EC50) of 2.65 mg l-1 after 48 h of exposure. The algae appeared to be at least 2 times less sensitive to EQ than fish embryos or daphnids. The results were used to calculate the risk for aquatic life resulting in a maximum tolerable level of 1 µg l-1 for fish embryos and daphnids, with a safety factor of 300. According to current knowledge, this does not exceed environmental concentrations of this substance. However, this study raises further concern about the (until recently) legal maximum tolerable EQ levels in fish feeding and the rather slow pace at which authorization to use EQ as a feed additive for diverse animals in Europe is being suspended.

Deoxynivalenol (DON) is a frequent contaminant of feeds in aquaculture, but the consequences of this contamination have rarely been evaluated. Previous studies on carp indicated effects of DON on liver function and histology after four weeks of feeding. The present study aimed to unravel the time course of liver responses of carp to orally applied DON. Therefore, liver enzyme activities and histology have been investigated after 7, 14, 26 and 56 days of DON feeding. The acute response comprises down-regulation of biotransformation enzymes, whereas the chronic response to DON is characterised by activation of alanine aminotransferase which indicates damage to liver tissue. Examination of histological sections of liver tissue revealed that changes such as fat aggregation, vacuolisation and hyperaemia were present after 14 and 26 days of exposure to DON but not thereafter. Several enzymes involved in glutathione cycling and reduction of oxidative stress were found to be reduced after 26 and 56 days of DON feeding. The results suggest that supporting the antioxidative system, e.g. by using glutathione-enriched yeast extracts as a food additive, might be successful in preventing the effects of DON in carp. This is the basis of a fundamental hypothesis since DON contamination of fish feed leads to pronounced effects on liver histology and liver enzyme activities which may also cause changes in the normal liver metabolism of endogenous and xenobiotic compounds.

The world-wide occurrence of zearalenone (ZEN) as a contaminant in feed for farm animals and fish requires the evaluation of toxicity mechanisms of action of ZEN. The present study investigates possible metabolization of ZEN in fish cell lines suggesting that mainly glucuronidation takes place. It demonstrates that concentrations up to 20,000 ng ml(-1) ZEN are capable of influencing cell viability in permanent fish cell cultures in a dose-response manner with different response patterns between the five tested cell lines, whereby lysosomes appeared to be the main target of ZEN. ZEN toxicity is often discussed in the context of oxidative stress. Our study shows a biphasic response of the cell lines when reactive oxygen species (ROS) production is monitored. Damage in cells was observed by measuring lipid peroxidation, DNA strand breaks, and alterations of intracellular glutathione levels. Metabolization of ZEN, especially at concentrations above 7500 ng ml(-1) ZEN, does not prevent cytotoxicity. ZEN as an estrogenic compound may involve processes mediated by binding to estrogen receptors (ER). Since one cell line showed no detectable expression of ER, an ER-mediated pathway seems to be unlikely in these cells. This confirms a lysosomal pathway as a main target of ZEN in fish cells.

Deoxynivalenol (DON) is one of the most important members of Fusarium toxins since it often can be found in relevant concentrations in animal feeds. The effects of this group of toxins on fish are mostly unknown. The present study shows results from a feeding trial with carp (Cyprinus carpio L.) using three different concentrations of DON (352 μg kg−1, 619 μg kg−1, and 953 μg kg−1 final feed, respectively) which are comparable to levels found in commercial fish feeds. Effects on growth and mass of fish were not observed during this 6 weeks lasting experiment. Only marginal DON concentrations were found in muscle and plasma samples. Blood parameters were not influenced although smaller erythrocytes occurred in fish treated with 352 μg kg−1 DON. Analysis of antioxidative enzymes in erythrocytes showed increased superoxid dismutase and catalase activities in fish fed the low-dose feed. Immunosuppressive effects of DON were confirmed whereby cytotoxic effects on immune cells only partly explained the impairment of innate immune responses. Exact polarization of the immune system into pro-inflammatory or anti-inflammatory responses due to DON exposure should be clarified in further experiments, especially since the current results raise concern about impaired immune function in fish raised in aquaculture.

The control of mycotoxins is a global challenge not only in human consumption but also in nutrition of farm animals including aquatic species. Fusarium toxins, such as deoxynivalenol (DON) and zearalenone (ZEN), are common contaminants of animal feed but no study reported the occurrence of both mycotoxins in fish feed so far. Here, we report for the first time the occurrence of DON and ZEN in samples of commercial fish feed designed for nutrition of cyprinids collected from central Europe. A maximal DON concentration of 825 μg kg−1 feed was found in one feed whereas average values of 289 μg kg−1 feed were noted. ZEN was the more prevalent mycotoxin but the concentrations were lower showing an average level of 67.9 μg kg−1 feed.

Zearalenone (ZEN) is a frequent contaminant of animal feeds, but systemic effects on fish and possible metabolic costs have not yet been investigated. In order to fill this gap a feeding trial with juvenile carp was conducted. The fish were fed ZEN-contaminated diets at three concentrations (low: 332 μg kg − 1 , medium: 621 μg kg − 1 , and high: 797 μg kg − 1 final feed, respectively) for four weeks. Possible reversible effects of ZEN were evaluated by feeding an additional group with the mycotoxin for four weeks period and the uncontaminated diet for further two weeks. After that possible ZEN effects on enzyme activities in kidney, spleen, liver and muscle tissue were investigated to get an organism-wide aspect of ZEN effects. Most organs appeared to (over)compensate ZEN effects during the exposure to this mycotoxin, which caused metabolic costs. Oxygen consumption increased in fish treated with the two higher ZEN concentrations via the diet. The differences between the treatments persisted also after the recovery phase of two weeks. Thus, the present study provided evidence of effects of ZEN on carbohydrate metabolism, lipid peroxidation in organs and metabolic oxygen demand. This is the first evidence for increased metabolic costs in a fish species due to exposure to the mycotoxin ZEN.

Contamination of animal feed with mycotoxins is a major problem for fish feed mainly due to usage of contaminated ingredients for production and inappropriate storage of feed. The use of cereals for fish food production further increases the risk of a potential contamination. Potential contaminants include the mycotoxin deoxynivalenol (DON) which is synthesized by globally distributed fungi of the genus Fusarium . The toxicity of DON is well recognized in mammals. In this study, we confirm cytotoxic effects of DON in established permanent fish cell lines. We demonstrate that DON is capable of influencing the metabolic activity and cell viability in fish cells as determined by different assays to indicate possible cellular targets of this toxin. Evaluation of cell viability by measurement of membrane integrity, mitochondrial activity and lysosomal function after 24 h of exposure of fish cell lines to DON at a concentration range of 0–3000 ng ml −1 shows a biphasic effect on cells although differences in sensitivity occur. The cell lines derived from rainbow trout are particularly sensitive to DON. The focus of this study lies, furthermore, on the effects of DON at different concentrations on production of reactive oxygen species (ROS) in the different fish cell lines. The results show that DON mainly reduces ROS production in all cell lines that were used. Thus, our comparative investigations reveal that the fish cell lines show distinct species-related endpoint sensitivities that also depend on the type of tissue from which the cells were derived and the severity of exposure.

In this study, carp Cyprinus carpio were injected with various steroid compounds, including synthetic and natural progestogens and the glucocorticoid cortisol, to investigate effects on leucocytes isolated from their kidneys. Injection of cortisol led to an increased spleeno-somatic index (I(S)) on day 21 post-injection (pi) and immunosuppressive effects measured as decreased nitric oxide (NO) production and increased arginase activity in isolated leucocytes on days 14 and 21 pi, respectively. Moreover, reduced NO production was also observed after injection of the synthetic progestogens, levonorgestrel (LEV) and medroxyprogesterone acetate. In addition, LEV influenced arginase activity in head kidney cells on day 14 and day 21 pi. This study is the first demonstration in fishes that the application of these steroid compounds in vivo affects NO production and arginase activity of isolated leucocytes.

A 56-day feeding trial was conducted to access the effect of housefly maggot meal (magmeal) diets on the performance, concentration of plasma glucose, cortisol and blood characteristics of Oreochromis niloticus fingerlings. Seven feeds formulated to contain 36% protein and 20 kJ g(-1) gross energy (dry matter basis), were prepared by replacing fish meal with magmeal. Fifteen fingerlings (initial average weight 2.0 +/- 0.1 g) stocked per experimental tank were fed in triplicates at 5% body weight in two portions per day (a level previously established). Growth and food conversion ratio were adequate and comparable without any significant differences (p < 0.5) between feeding groups. Mean values for haematocrit and plasma glucose were not significantly different (p < 0.05) among the feeding groups. Fish group fed control diet (containing highest inclusion level of fish meal and without magmeal) gave the lowest haemoglobin concentration (5.96 +/- 0.22 g dl(-1)). This value was significantly different from other feeding groups. Stressful conditions in fish and in mammals are associated with decreased growth, haematocrit (packed cell volume) and haemoglobin values, increased whole blood glucose (hyperglycaemia) and plasma cortisol concentrations. No such physiological changes were observed in this study. Results suggest that feeding O. niloticus fingerling with magmeal diets did not cause any form of physiological stress. Magmeal can be used as a good alternative protein source in tilapia diets.

The inducible nitric oxide synthase (iNOS) plays a central role in the inflammatory reactions that follow infection or tissue damage. Induction of nitric oxide (NO) synthesis by bacterial lipopolysaccharide (LPS) depends on activation of G protein-coupled receptors in mammals. Thus, it was our intention to evaluate whether similar mechanisms are involved in iNOS activation in fish leukocytes. Therefore, the participation of membrane-bound receptors which activate effectors via G proteins has been confirmed using the G protein inhibitor suramin. Furthermore, the NO produced by iNOS performs both beneficial and detrimental actions. It is thus conceivable that regulatory mechanisms exist which control the timing and intensity of NO production by iNOS in order to outweigh protective effects against detrimental ones. The second messenger cAMP produced by adenylyl cyclases (ACs) plays a key role in the regulation of many cellular functions. Since cAMP signaling inhibits numerous immunological reactions, studies have been carried out to determine whether cAMP-dependent pathways could inhibit NO production by carp leukocytes as well. To measure cellular responses such as NO production by carp leukocytes derived from head and trunk kidneys treatments were performed with the cAMP elevating agents forskolin and dibutyryl-cAMP (db-cAMP) prior to stimulation with Aeromonas hydrophila. Pharmacological studies in stimulated kidney leukocytes showed that increased intracellular cAMP levels lead to reduced NO formation. This reduction of NO production was not due to decreased cell numbers, since a tetrazolium dye-based assay revealed no reduction of cell viability by cyclic nucleotide elevating agents. Thus, our data provide evidence that the AC/cAMP signaling pathway is well established in carp leukocytes. Cyclic AMP leads to type II immune response. We provide evidence that the predominant AC in fish leukocytes is a particulate enzyme due to its sensitivity to forskolin. Treatment of leukocytes with agents increasing intracellular cAMP gave clear evidence for participation of this cyclic nucleotide in immune signaling.

The mycotoxin zearalenone (ZEN) is frequently contaminating animal feeds including feed used in aquaculture. In the present study, the effects of dietary exposure to ZEN on carp (Cyprinus carpio L.) were investigated. ZEN at three different concentrations (low dose: 332 µg kg(-1), medium dose: 621 µg kg(-1) and high dose: 797 µg kg(-1) final feed, respectively) was administered to juvenile carp for four weeks. Additional groups received the mycotoxin for the same time period but were fed with the uncontaminated diet for two more weeks to examine the reversibility of the ZEN effects. No effects on growth were observed during the feeding trial, but effects on haematological parameters occurred. In addition, an influence on white blood cell counts was noted whereby granulocytes and monocytes were affected in fish treated with the medium and high dose ZEN diet. In muscle samples, marginal ZEN and α-zearalenol (α-ZEL) concentrations were detected. Furthermore, the genotoxic potential of ZEN was confirmed by analysing formation of micronuclei in erythrocytes. In contrast to previous reports on other fish species, estrogenic effects measured as vitellogenin concentrations in serum samples were not increased by dietary exposure to ZEN. This is probably due to the fact that ZEN is rapidly metabolized in carp.

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch), The mycotoxin deoxynivalenol (DON) has been shown to regularly occur at relevant concentrations in feed designed for aquaculture use, but little is known about the consequences of its presence on the organisms that consume the DON-contaminated feed. Previous studies indicated a down-regulation of pro-inflammatory responses in carp (Cyprinus carpio L.) after 4 weeks of feeding DON. The present study examined the time course of innate immune responses of carp to orally administered DON. Changes in mRNA levels of immune genes in different organs (head kidney, trunk kidney, spleen, liver, and intestine) were observed indicating immune-modulating properties of DON. The immune-modulatory effects during the acute phase of DON exposure were characterized by the activation of both pro- and anti-inflammatory cytokines and enzymes in carp. The subchronic responses to DON were characterized by activation of arginases culminating in increased arginase activity in head kidney leukocytes after 26 days of DON treatment. These results suggest profound effects of this mycotoxin on fish in aquaculture.

The mycotoxin zearalenone (ZEN) is a frequent contaminant of animal feeds, but its effects on fish have not yet been investigated extensively. In order to fill this gap a feeding trial with juvenile carp was conducted. Three groups of fish were fed feeds contaminated with ZEN at three concentrations (low: 332 μg kg−1, medium: 621 μg kg−1, and high: 797 μg kg−1feed) for four weeks. Possible reversible effects of ZEN were evaluated by feeding additional groups with the ZEN-contaminated feeds for four weeks, followed by the uncontaminated diet for two weeks. Immune function of isolated leukocytes from head kidney and trunk kidney was assessed using the assessment of NO production, the respiratory burst assay, the chemiluminescence assay, and the measurement of arginase activities. These investigations frequently revealed increased immune responses after exposure of fish to low ZEN concentrations and reduced immune responses after exposure to high mycotoxin concentrations. Moreover, the feeding of the uncontaminated diet for further two weeks did not improve the immune responses in most cases. These results indicate that cellular immune functions in ZEN-contaminated carp are influenced which may be relevant for fish health in aquaculture.

Dissolved natural organic matter (NOM) is dead organic matter exceeding, in freshwater systems, the concentration of organic carbon in all living organisms by far. 80-90% (w/w) of the NOM is made up of humic substances (HS). Although NOM possesses several functional groups, a potential effect on aquatic organisms has not been studied. In this study, direct effects of NOM from various origins on physiological and biochemical functions in the aquatic plant Ceratophyllum demersum are presented. Environmentally relevant concentrations of NOM cause inhibitory effects on the photosynthetic oxygen production of C. demersum. Various NOM sources and the synthetic humic substance HS1500 inhibit the photosynthetic oxygen production of the plant as observed with 1-amino-anthraquinone, a known inhibitor of plant photosynthesis. 1-Aminoanthraquinone may serve as an analogue for the quinoid structures in NOM and HS. Most likely, the effects of NOM may be related to quinoid structures and work downstream of photosynthesis at photosystem (PS) II.

Deoxynivalenol (DON) frequently contaminates animal feed, including fish feed used in aquaculture. This study intends to further investigate the effects of DON on carp (Cyprinus carpio L.) at concentrations representative for commercial fish feeds. Experimental feeding with 352, 619 or 953 μg DON kg(-1) feed resulted in unaltered growth performance of fish during six weeks of experimentation, but increased lipid peroxidation was observed in liver, head kidney and spleen after feeding of fish with the highest DON concentration. These effects of DON were mostly reversible by two weeks of feeding the uncontaminated control diet. Histopathological scoring revealed increased liver damage in DON-treated fish, which persisted even after the recovery phase. At the highest DON concentration, significantly more fat, and consequently, increased energy content, was found in whole fish body homogenates. This suggests that DON affects nutrient metabolism in carp. Changes of lactate dehydrogenase (LDH) activity in kidneys and muscle and high lactate levels in serum indicate an effect of DON on anaerobic metabolism. Serum albumin was reduced by feeding the medium and a high dosage of DON, probably due to the ribotoxic action of DON. Thus, the present study provides evidence of the effects of DON on liver function and metabolism.

Worldwide increases in fluvial fine sediment are a threat to aquatic animal health. Fluvial fine sediment is always a mixture of particles whose mineralogical composition differs depending on the sediment source and catchment area geology. Nonetheless, whether particle impact in aquatic organisms differs between mineral species remains to be investigated. This study applied an in vitro approach to evaluate cytotoxicity and uptake of four common fluvial mineral particles (quartz, feldspar, mica, and kaolin; concentrations: 10, 50, 250 mg L(-1)) in the rainbow trout epithelial gill cell line RTgill-W1. Cells were exposed for 24, 48, 72, and 96 h. Cytotoxicity assays for cell membrane integrity (propidium iodide assay), oxidative stress (H2DCF-DA assay), and metabolic activity (MTT assay) were applied. These assays were complemented with cell counts and transmission electron microscopy. Regardless of mineral species, particles ≤ 2 µm in diameter were taken up by the cells, suggesting that particles of all mineral species came into contact and interacted with the cells. Not all particles, however, caused strong cytotoxicity: Among all assays the tectosilicates quartz and feldspar caused sporadic maximum changes of 0.8-1.2-fold compared to controls. In contrast, cytotoxicity of the clay particles was distinctly stronger and even differed between the two particle types: mica induced concentration-dependent increases in free radicals, with consistent 1.6-1.8-fold-changes at the 250 mg L(-1) concentration, and a dilated endoplasmic reticulum. Kaolin caused concentration-dependent increases in cell membrane damage, with consistent 1.3-1.6-fold increases at the 250 mg L(-1) concentration. All effects occurred in the presence or absence of 10% fetal bovine serum. Cell numbers per se were marginally affected. Results indicate that (i.) natural mineral particles can be cytotoxic to gill epithelial cells, (ii.) their cytotoxic potential differs between mineral species, with clay particles being more cytotoxic, and (iii.) some clays might induce effects comparable to engineered nanoparticles.

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch), The worldwide distribution and high bioaccumulation potential of pentachlorophenol (PCP) in aquatic organisms imply a high toxicological impact in aquatic systems. Firstly, our investigations show that, similar to mammalian cell lines, PCP can be metabolized to tetrachlorohydroquinone (TCHQ) in the permanent cell line derived from rainbow trout liver cells (RTL-W1). Moreover, we demonstrate that PCP as well as its metabolite TCHQ is capable of influencing the viability of these cells. Three cell viability assays were performed to assess possible cellular targets of these substances. Thus, the cytotoxicity of the PCP-derivative TCHQ was shown for the first time in a fish cell line. Further investigations revealed the involvement of ROS in the cytotoxicity of PCP and its metabolite TCHQ. The observation of oxidative stress provides a plausible explanation for the increased cytotoxicity at higher concentrations especially for PCP and implies possible mechanisms underlying these observations. In addition, antioxidants such as ascorbic acid and quercetin modulate the detrimental effects of PCP and TCHQ whereby both compounds exacerbate the cytotoxic effects of high PCP and TCHQ concentrations.

In an aquatic ecosystem, during cyanobacterial bloom lysis, a mixture of toxins and other cyanobacterial and bacterial components will be present in the water, acting on aquatic organisms. Most of the research into toxic effects of cyanobacteria has involved the use of purified toxins. In this study, the "real-life" situation of a cyanobacterial lysis event was investigated. For this purpose, intact cells from a natural cyanobacterial bloom from Lake Muggelsee, Berlin, were taken and the cells were broken by repeated freeze/thaw cycles. This crude extract was used to expose several aquatic organisms ranging from microalgae (Scenedesmus armatus), macrophyte (Ceratophyllum demersum), invertebrate (Chaoborus crystallinus) up to fish eggs (Danio rerio) to look at several physiological parameters such as detoxication enzyme activity and, in the case of the microalgae and the macrophyte, also the effect on activity of photosynthesis. In all the tests, the cyanobacterial crude extract caused stronger effects than the pure cyanobacterial toxins used in equivalent concentrations.

Environmental compounds can interfere with endocrine systems of wildlife and humans. The main sink of such substances, called endocrine disrupters (ED), are surface waters. Thus, aquatic vertebrates, such as fish and amphibians, are most endangered. ED can adversely affect reproductive biology and the thyroid system. ED act by (anti)estrogenic and (anti)androgenic modes of action, resulting in abnormal sexual differentiation and impaired reproduction. These effects are mainly driven by direct interferences of ED with sex steroid receptors rather than indirectly by impacting synthesis and bioavailability of sex steroids, which in turn might affect the hypothalamic-pituitary-gonadal axis. Recent findings reveal that, in addition to the human-produced waste of ED, natural sources, such as parasites and decomposition of leaves, also might act as ED, markedly affecting sexual differentiation and reproduction in fish and amphibians. Although the thyroid system has essential functions in both fish and amphibians, amphibian metamorphosis has been introduced as the most sensitive model to detect thyroidal ED; no suitable fish model exists. Whereas ED may act primarily on only one specific endocrine target, all endocrine systems will eventually be deregulated as they are intimately connected to each other. The recent ecotoxicological issue of pharmaceutically active compounds (PhACs) present in the aquatic environment indicates a high potential for further endocrine modes of action on aquatic vertebrates by ED derived from PhACs, such as glucocorticoids, progestins, and beta-agonists.

The involvement of steroid hormones in direct and indirect regulation and modulation of immune responses is well recognized in mammals. Here, we demonstrate that progestogens are capable of influencing the innate immunity in fish as well. Therefore, we confirmed the known immunosuppressive effects of natural progesterone (P4), and compared them to influences of 17α,20β-dihydroxy progesterone (DHP4) and the synthetic progestins, medroxyprogesterone acetate (MPA) and levonorgestrel (LEV), on NO release by in vitro-stimulated carp leukocytes derived from both, head and trunk kidney, respectively. DHP4 known as the main maturation-inducing steroid in many teleosts potently inhibited the NO release by carp leukocytes. The synthetic progestin MPA, which may also be environmentally relevant due to its world-wide use in hormonal contraception, significantly decreased NO formation by head and trunk kidney cells. In contrast, LEV showed no significant influence on NO release by head and trunk kidney leukocytes. The observed immunosuppressive actions of progestogens on NO production were compared to the known impairment by natural and synthetic glucocorticoids. Determining the potential impact of progestogens on mRNA expression of iNOS by means of semi-quantitative reverse transcription polymerase chain reactions (RT-PCR) revealed downregulation of proinflammatory type I immune response characteristics at high concentrations. These findings demonstrate for the first time that similar to the known effects of natural progesterone synthetic progestogens are also able to influence immune signaling cascades in fish, and provide evidence that these steroids are capable of influencing mRNA expression of iNOS. The induction of a regulatory type II immune response by progestogens is a striking example of interference of female steroid-mediated events with the piscine immune system. Furthermore, the identification of a partial sequence of a membrane-associated progestogen receptor (mPR) in carp leukocytes by RT-PCR indicates a specific mechanism underlying the observed effects of progestogens on these immune cells.